There is an interest in visualization of neurotransmission for optical imaging of metabolic and signaling enzymes in cells and tissues. [1, 2]
Dopaminergic neurotransmission plays key roles in motivational behavior, reward and habit learning, working memory and cognition, while aberrations in presynaptic dopamine stores and release underlie important aspects of psychiatric disorders including schizophrenic and amphetamine-triggered psychosis, ADHD and drug addiction as well as Parkinson's disease and methamphetamine toxicity.
The termination of neurotransmitter action is determined by a number of factors, including their reuptake into nerve terminals by monoamine transporters, their dilution by diffusion out of the synaptic cleft, and their metabolism by Monoamine Oxidase. Specific monoamine transporters located in the neuronal plasma membrane terminate the action of neurotransmitters by transporting them back into presynaptic terminals. Once inside the presynaptic terminal, vesicular monoamine transporters mediate their filling into secretory vesicles. All characterized monoaminergic cells utilize the vesicular monoamine transporter (VMAT) to accumulate monoamines from the cytoplasm into vesicles. These VMATs are polytopic membrane proteins, which act as electrogenic antiporters (exchangers) of protons and monoamines utilizing an acidic and positively polarized granule matrix.
The monoamine transporters of synapses formed by the midbrain dopamine projections are involved in voluntary motor control, reward and learning, and are the primary target of drugs of abuse including amphetamine, nicotine, cocaine as well as therapeutic agents that are used to treat mood disorders. Neuronal death in the substantia nigra is the cause of Alzheimer's disease and a decreased density of dopamine monoamine transporter has been found in Parkinson's, Wilson's, and Lesch-Nyhan's disease, while a decrease in serotonin monoamine transporter level is found in patients suffering from major depression and aggressive behavior.
Fluorescent false neurotransmitters (FFNs), probes that act as optical tracers of dopamine and provide the first means to image neurotransmitter release from individual presynaptic terminals in the brain have recently been introduced. [3] FFNs have been designed to act as tracers of dopamine to enable direct visualization of neurotransmitter uptake and release at individual synaptic terminals. FFNs represent a novel class of imaging probes that are small organic compounds that fulfill the following criteria: (a) like dopamine, they accumulate into synaptic vesicles and chromaffin vesicles in a manner dependent on the vesicular monoamine transporter (VMAT) and pH gradient; (b) like dopamine, they are released with vesicle fusion; (c) unlike dopamine, they are intensely fluorescent so that they can be used in low concentrations to avoid interference with normal transmitter release; (d) they are non-toxic and photostable, limiting bleaching. FFNs are described in PCT/US2007/017014 (WO 2008/013997), which is hereby incorporated by reference in its entirety.
A non-fluorescence-based technology, Positron Emission Tomography (PET), enables CNS imaging in live animals and humans and is widely used in preclinical and clinical research, drug development and medicine. FFNs incorporating radioactive fluorine isotope F18 for use in PET imaging are described in PCT/US2009/000630 (WO 2009/097144), which is hereby incorporated by reference in its entirety.
Monoamine neurotransmitters are accumulated in synaptic vesicles by vesicular monoamine transporter 2 (VMAT2), which translocates the monoamine (e.g. dopamine) from cytosol to the lumen of synaptic vesicles. [5] Similarly, in chromaffin cells of adrenal medulla, epinephrine is accumulated in secretory vesicles by a closely related protein VMAT1 (FIG. 1). The vesicular lumen is acidic (pH˜5-6) due to the action of vacuolar-H+ ATPase, which imports H+ at the expense of ATP hydrolysis. The pH gradient between the cytoplasm and the vesicular lumen in turn provides the driving force for the accumulation of neurotransmitters in the vesicles. Thus the pH gradient is one of the key parameters regulating synaptic plasticity as it controls the vesicle content and potentially the size of the releasable pool.
The pH gradient between the cytosol and the vesicular lumen is ATP dependent and thus closely coupled to the metabolic state of the presynaptic terminals. Despite the importance of this parameter there are currently no small molecule probes available for selective measurement of pH in synaptic or secretory vesicles. The pH-sensitive synaptopHluorin protein has been developed to measure pH in synaptic vesicles of cultured neurons. [6] Alternatively, construction of avidin-chimera proteins allow for anchoring a pH sensitive fluorescent dye, linked to biotin, to specific organelles including secretory vesicles. [7] These approaches, however, require transfection of the cell culture prior to measurement, or generation of transgenic animals for studies with tissue.
Commercially available pH-sensitive dyes (e.g. Lysotracker) [8] are not suitable for this task as they label other acidic organelles including endosomes and lysosomes, and in the brain they do not label presynaptic terminals. [9] Moreover, such dyes cannot measure pH within the synaptic vesicles in the brain.
Pancreatic β-cells, just like chromaffin cells, contain VMAT on the plasma membrane of their secretory vesicles. [C11]DTBZ or fluoropropyl-9-desmethyl-DTBZ are used to determine the amount of VMAT, thus enabling researchers to determine the amount of pancreatic β-cells. But neither [C11]DTBZ nor fluoropropyl-9-desmethyl-DTBZ are suitable for imaging dynamic changes in transmitter pools.
Herein, pH responsive compounds and their use in optical in situ measurement of pH, including pH changes, are described.